Softworx deconvolution software

Indirect immunofluorescence assays (IFA) were performed on freshly collected tachyzoites washed with buffer A with glucose (BAG, 116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO₄, 50 mM HEPES, pH 7.2, and 5.5 mM glucose) and fixed with 4% formaldehyde for 1 h, permeabilized with 0.3% Triton X-100 for 20 min, and blocked with 3% bovine serum albumin (Miranda et al., 2010 (link)). Immunofluorescence was performed as previously described (Miranda et al., 2010 (link)) and primary and secondary antibodies concentrations are indicated in the figure legends. Fluorescence images were collected with an Olympus IX-71 inverted fluorescence microscope with a Photometrix CoolSnapHQ CCD camera driven by DeltaVision software (Applied Precision, Seattle, WA). Collected images were deconvolved using Softworx deconvolution software (Applied Precision, Seattle, WA). For all images, 15 cycles of enhanced ratio deconvolution were used.
Western blot analysis was performed as previously described (Miranda et al., 2010 (link)). We used the anti-TgVP1 polyclonal antibody at a dilution of 1:500 and secondary goat anti-Guinea pig antibody conjugated with HRP at 1:5,000. Mouse anti-α-tubulin at a dilution of 1:1,000 was used as a loading control.

Z-stack images were collected with a DeltaVision wide field fluorescence microscope (Applied Precision, GE) equipped with a digital camera (CoolSNAP HQ; Photometrics) and a 1.4-numerical aperture 100× objective lens. Excitation light was generated with an Insight SSI solid state illumination module (Applied Precision, GE), and the images were deconvolved with SoftWoRx deconvolution software (Applied Precision, GE). In all experiments, identical acquisition conditions were used to acquire all images. Following deconvolution, images were analyzed using Imaris 8.4.1 (Bitplane). An algorithm was designed using the surface function in Imaris to generate surfaces around the signal of interest and the maximum fluorescence intensity detected within these surfaces was measured. The same algorithm was applied to all images within an experiment. For live cell experiments, cells were plated on delta DPG dishes (Thermo Fisher Scientific) and maintained at 5% CO₂ and 37 °C in an environmental chamber on a DeltaVision microscope.